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Electro-spinning of PLGA/PCL blends for tissue engineering and their

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Article  in  Journal of Materials Science: Materials in Medicine · March 2010

DOI: 10.1007/s10856-010-4048-y · Source: PubMed

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J Mater Sci: Mater Med
DOI 10.1007/s10856-010-4048-y
Electro-spinning of PLGA/PCL blends for tissue engineering
and their biocompatibility
Nguyen Thi Hiep • Byong-Taek Lee
Received: 30 November 2009 / Accepted: 1 March 2010
Ó Springer Science+Business Media, LLC 2010
Abstract In this study, an electro-spun co-polymer
PLGA/PCL blend was fabricated using various percentages
of PLGA in the blend PLGA/PCL solutions. The PLGA/
PCL ratios used to fabricate the electrospun fibrous mats
were reflected in the FT-IR (Fourier Transform Infrared
Spectroscopy) data. Experimental results from the MTT
assay showed that the biocompatibility of the electro-spun
co-polymer increased at increasing percentages of PLGA.
In vitro cells adhesion and proliferation of fibroblast cells
on electro-spun mats were characterized by SEM mor-
phology. In addition, we found that increasing PLGA
concentrations affected the mechanical properties of elec-
tro-spun membranes and increased the biocompatibility of
PLGA/PCL electro-spun fibrous mats.
1 Introduction
Tissue engineering is an emerging multidisciplinary field
involving biology, medicine, and engineering and this field
is likely to revolutionize the ways we improve health by
restoring, maintaining, or enhancing tissue and organ
function [1]. The foundation of tissue engineering for
diagnostic applications is based on the ability to exploit
living cells in a variety of ways. Tissue engineering
research includes the following areas: biomaterials, cells,
biomolecules, engineering design aspects, biomechanical
aspects of design, informatics to support tissue engineering,
stem cell research, etc. [2]. In terms of biomaterials, novel
N. T. Hiep
B.-T. Lee (&)
Department of Biomedical Engineering and Materials,
College of Medicine, Soonchunhyang University,
Cheonan 330-090, Korea
e-mail: lbt@sch.ac.kr
biomaterials have been designed to direct the organization,
growth, and differentiation of cells in the process of
forming functional tissue by providing both physical and
chemical cues [3]; The involvement of cells in the tissue
engineering field include developing novel methods for the
proliferation and differentiation of cells. The biomechani-
cal aspects of tissue engineering design includes properties
of native tissues, identification of minimum properties
required for engineered tissues, mechanical signals regu-
lating engineered tissues, and efficacy and safety of engi-
neered tissues [4].
The electro-spinning method is one of the simplest
among all methods for the preparation of fibrous mats as
extra-cellular matrices. Electro-spinning offers a new
direction for biopolymers since this approach allows one to
decrease the diameter of the pore size from micrometers to
nanometers; thereby, increasing the surface area to volume
or mass ratio and the mechanical properties of the polymer
[5]. The advantages of the electro-spinning technique are
the production of very thin fibers, on the order of few
nanometers or micrometers, with large specific surface
areas, which allow for easy functionalization for various
applications, superior mechanical properties and ease of
processing, as suggested by many experts in the tissue
engineering field [6]. Electro-spun scaffolds allow cells to
grow while providing sufficient mechanical support. Tan
et al. reported that the mechanical properties of fibers
increased when the inner diameter of the fiber was
decreased [7]. The scaffolds currently used for arteries,
however, are still far from optimal in terms of mechanical
endurance and biocompatibility. The structure and prop-
erties of electro-spun biopolymer scaffolds are of critical
importance for their capable application in tissue engi-
neering. It has been generally accepted that the electrospun
mats need to be biocompatible, biodegradable with
123

degradation or resorbable rates that match the tissue
replacement and possess good mechanical properties to
match those of the tissues at the site of implantation. The
characteristics of electrospun mats include an appropriate
extracellular matrix structure to facilitate cell adhesion,
proliferation and differentiation, architecture similar to a
real tissue made from biodegradable or biocompatible
material, an internal architecture with fully interconnected
pores and mechanical strength that satisfies physiological
requirements. In recent years, many polymers have
undergone electro-spinning to fabricate nano-fibers for
different applications such as artificial skin [8], artificial
blood vessels [9], bone tissue engineering [10], bladder,
etc. The polymer combinations used in these processes
have included the use of a single natural polymer [11],
synthetic polymer [12], copolymer consisting of a natural
and synthetic polymer [13], etc. However, to overcome the
limitation associated with a single polymer, a new method
was developed that involves the simultaneous electro-
spinning of a blend of two polymer solutions delivered by
mixing the two polymer then electro-spinning. The blend
electro-spun method not only overcomes the limitations
associated with a single polymer, but also creates a
new component without the need for synthesizing a new
co-polymer, which is difficult and complex to achieve
using co electro-spinning of two single biopolymers. In the
blend electro-spun method, one can blend the two polymers
using an electro-spun 2-eject system, which contains a mix
of the polymers and then electro-spin the mix. For exam-
ple, Duan et al. [8] investigated electro-spun PLGA–PVA/
chitosan blends for skin applications, where they mixed
PVA and chitosan during the first electro-spinning injection
and PLGA was added during the second electro-spinning
injection. Using this approach, they obtained PLGA-PVA/
chitosan mats that had the strong mechanical properties of
PLGA, absorptive properties of PVA and antibacterial
activity of chitosan.
In this research, electro-spun PLGA/PCL mats were
fabricated from PLGA (85:15), which is an expensive
synthesis polymer. Therefore, PCL was used as the main
component because it is relatively inexpensive and stable
in material handling and storage [14]. PCL is a flexible
biopolymer that has been used to overcome the brittle and
low elongate properties of PLGA [15]. However, PLGA is
better than PCL in respect to cell adhesion and proliferation
due to its more hydrophilic property [15]. Polycaprolactone
(PCL) is hydrophobic and, thus, does not have any physi-
ological active sites, which makes PCL unfavorable for cell
growth when it comes into contact with the living body.
Although, when the behavior of epithelial cells was
evaluated on a PCL membrane, the PCL membrane was
shown to enhance epithelium growth. However, cell
affinity towards PCL is generally poor due to their low
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J Mater Sci: Mater Med
hydrophilicity and lack of surface cell recognition sites
[16]. Even though the electrospun PCL mats mimic the
identity of ECM in living tissues, its poor hydrophilicity
caused a reduction in cell adhesion, migration, prolifera-
tion, and differentiation [17, 18]. The advantage of using
PCL in this composite is PCL has high mechanical
strength, which is a high valuable property for tissue
engineering applications. PLGA was added to the com-
posite to support cells attachment, adhesion and cells
proliferation on the PLGA/PCL electro-spun mats, which
was previously demonstrated for human mesenchymal
stem cells and osteoblast cells [19, 20]. The morphology of
the fine PLGA/PCL electro-spun fibrous mats was imaged
by SEM. The composition and mechanical properties of
the blend electrospun fibrous mats were characterized by
FT-IR and tensile strength tests, respectively. In vitro cell
adhesion and proliferation were tested by seeding cells for
60 min, 1 day and 3 days. The MTT assay showed that the
PLGA/PCL electrospun mats were highly biocompatibility.
2 Materials and methods
2.1 Materials
PLGA (85:15), polycaprolactone (Mn 80 000), tetrahydro-
furan (THF, minimum 99%), Dimethylformamide (DMF,
99%) were purchased from Sigma-Aldrich, USA. Methyl
Cloride (MC, SK Chemicals Co., Korea). DMSO
(Dimethylsulfoxide 99, 0%, Samchun Pure Chemical Co.,
LTD, Korea), Ethanol (Merck, Germany). Fetal bovine
serum (FBS), P.S. (penicillin/streptomycin [antibiotics]),
Dulbecco’s phosphate buffered saline (D-PBS) without
calcium or magnesiumand, MTT solution and trypsin–
EDTA were purchased from GIBCO (Carlsbad, CA). The
L-929 cells line was obtained from the ATCC Cell Line
(CCL-1TM, NCTC clone 929 [L cell, L-929, derivative of
Strain L], Korea). HDMS (Hexamethyldisilazane, Sigma,
USA).
2.2 Preparation of polymer solutions
10 wt% PLGA was dissolved in THF: DMF (50:50) [8] and
12 wt% PCL was dissolved in MC: DMF (20:80) [21].
Both solutions were prepared overnight until the solution
became homogeneous. Then, several PLGA/PCL azeotrope
solutions were prepared by adding a homogeneous PLGA
solution to a homogeneous PCL solution.
2.3 Electro-spinning setting
PLGA/PCL blend solutions were placed in a plastic syringe
(lure-lock type, 12 ml) and connected through a metal
J Mater Sci: Mater Med
syringe needle, which was already connected to a nozzle
gauge (25 gauge, inner diameter 0.25 mm) on the pump.
The parameters of electrospinning were as follows: the
solution flow rate was 0.5 ml/h, a high voltage of 25 kV
was supplied directly from a high DC voltage power supply
(NNC-30 kilovolts-2 mA portable type, NanoNC Korea)
and the samples were collected through a steel plate 20 cm
away from the tip of the syringe needle.
2.4 Characterization
2.4.1 Morphology
The detailed structure of each polymer fibrous mat was
imaged by scanning electron microscopy (SEM) (SM-65F,
JEOL, and Japan) at an acceleration voltage of 25 kV. Before
SEM observation, all of the samples cut from the electrospun
fiber mats (1 cm 9 1 cm) were sputter coated with gold
under a JEOL JFC-1200 fine coater for 60 s. The average
fiber diameter of the electrospun fibers was measured from
the SEM images using Adobe Photoshop 5.0 software.
2.4.2 FT-IR analyze
The electro-spun PCL, PLGA and blend PLGA/PCL nano-
fibrous mats were characterized by attenuated reflectance
Fourier transform spectroscopy (Spectrum GX, PerkinEl-
mer, USA). The infrared spectra of the samples were
measured over a wavelength range of 4,000–500 cm-1. All
spectra were taken in the spectral range by the accumula-
tion of 64 scans with a resolution of 4 cm-1.
2.4.3 Mechanical properties
The dimensions of the samples were measure by SEM
before measuring the tensile strength (thick ness 100 lm,
wide 1 mm and length 27 mm). Mechanical characteriza-
tion was achieved by applying tensile test loads to speci-
mens prepared from the electro-spun ultra fine non-woven
fiber mats. Since a single polymer nano-fiber is very weak,
the resulting non-woven mat was so delicate that any direct
contact with the mat surface during manipulation could
damage the fibers. Thus, sufficient care was taken in pre-
paring and handling the tensile specimens in order to avoid
severe damage. The specimens were prepared using the
following method. First, a white paper frame was cut as
shown in Fig. 4, and sample (1) was glued (2) onto the top
and bottom areas of one side. The double-sided tape (3)
was 2 mm and the hold (4) was used to remove the frame
from the load-cell. The frame was then glued onto the
topside of the fiber mat, and was cut into rectangular pieces
along the vertical lines.
2.5 In vitro study
2.5.1 Sterilization approach and preparation sample
for in vitro
All electro-spun mats were placed in a vacuum oven for
48 h at room temperature and sterilized using 70% EtOH
for 30 min before conducting in vitro testing.
2.5.2 Cell line and maintenance
Cell culture studies were carried out with the L929 cell
line obtained from ATCC Cell Line (CCL-1TM, NCTC
clone 929 [L cell, L-929, derivative of Strain L], Korea).
The L-929 cells were subcultured in flasks using RPMI
media, supplemented with 10% (v/v) FBS (fetal bovine
serum) and 1% PS (penicillin/streptomycin (antibiotics)),
50 lg/ml ascorbic acid (Sigma) and 10 mM b-glycero-
phosphate (Sigma) at 37°C, 95% humidity and 5% CO2
(Incubator, ASTEC, Japan). Cells were dissociated with
trypsin–EDTA (GIBCO) centrifuged and resuspended in
medium. The culture medium was replaced every 2 days.
Prior to cell culture experiments, 24-well or 96-well tis-
sue culture test plates gamma sterilized (SPL Life Sci-
ence, Becton-Dickinson Labware) were prepared. The
culture plates were then sprayed with 70% ethanol before
being placing under UV light for 30 min to sterilize
them.
2.5.3 MTT assay/cytotoxicity assay
The cell samples were washed in PBS 1 time and medium 2
times before the cells were seeded. The cellular viability %
of fibroblast cells on each polymer fibrous mat was deter-
mined using the Cytotoxicity or MTT (3-[4, 5-dimethyl-
thiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assays.
The L-929 mouse fibroblasts cells were seeded in 96-well
tissue culture plates at 1,000 cells/well in 100 ll RPMI.
All media contained 10% FBS and all cell lines were
incubated overnight. Diluted extract solutions (100 ll) of
every fibrous mat at various concentrations (100, 75, 50, 25
and 0%) were then added. The L 929 fibroblast cells were
treated for 1, 2, and 3 days and then 20 ll of filtered MTT
solution (5 mg/mL in PBS) was added. After incubation at
37°C for 3.5 h, the medium was removed from the well and
150 ll of DMSO was added to dissolve any insoluble
formazan crystals. The absorbance was measured at
560 nm using an ELISA reader (Turner Biosystems CE,
Promega Corporation, USA) and the cell viability was
calculated as the percentage relative to the untreated con-
trol cells.
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2.5.4 Adhesion, growth behavior of cells
Sample membranes 20 mm in diameter were sterilized
with 70% EtOH (30 min), then washed with PBS and
suspended in conditioning medium (15 min). The fibro-
blast cells (L-929) were cultured on TCPs (Tissue Culture
Polystyrene), control-like and samples. The cells were
seeded at a cell density of 104 cells/cm2 in RPMI and
cultured for 1 and 3 days at 37°C in a humidified air
atmosphere with 5% CO2. After 1 and 3 days of culture,
cellular constructs were harvested, rinsed twice with PBS
to remove non-adherent cells and subsequently fixed with
2% glutaraldehyde for 15 min and washed with DPBS two
times (15 min/time). The samples were then dehydrated
through a series of graded ethanol (EtOH) solutions (50,
75, 90, 95 and 100%) and 5 min was used for every
washing time. The cells were then washed three times in
100% EtOH. Finally, the samples were washed two times
with HDMS. All samples were air-dried overnight. Dry
cellular constructs were sputtered with gold and imaged by
SEM.
3 Results
3.1 SEM morphology of co-polymer PLGA/PCL
electro-spun
The morphology of electro-spun ultra-fine fibers is influ-
enced by various parameters such as the applied voltage,
solution flow rate, distance between capillary and collector,
and especially the properties of the polymer solutions
Fig. 1 SEM morphology of the PLGA/PCL blend (10/90) (a), (20/80) (b), (30/70) (c) electrospun nano-fibrous mats
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J Mater Sci: Mater Med
including concentration, surface tension and the nature of
the solvents [22]. However, the solubility of the polymer
should be examined in more detail when the electro-spun
material is a co-polymer because the uniform electro-spun
material just appears as a homogenous dissolution. In this
study, we mixed several ratios of PLGA and PCL (10, 20
and 30% PLGA) that were from separate homogenous
solutions. The mixed polymers were incubated together for
6 h at room temperature to create a homogenous azeotrope.
The electro-spun PLGA/PCL mats were then fabricated
using an electro-spinning machine (NNC-30 kV-2 mA
portable type, Nano NC. Korea.). The parameters used for
the fabrication of each polymer fibrous mats were as fol-
lows: voltage 25 kV, distance 20 cm, flow rate 0.5 ml/h
and nozzle 25 G. SEM images and the diameter distribu-
tions of each different type of fiber are shown in Fig. 1a, b
and c. Based on the analysis of the SEM images, the
electro-spun mats were homologous and uniform over a
large area. However, morphology of each sample showed
differences in fibers; for example, in cases of PLGA/PCL
(10/90) (Fig. 1a), fibers were not cylindrical and were
attached to each other. Contrary to Fig. 1a, the case of
PLGA/PCL (20/80) (Fig. 1b) showed that electro-spun
fibers were cylindrical and separated with continuous
fibers. On the other hand, when the concentration of PLGA
was increased to 30, PLGA/PCL (30/70) (Fig. 1c) showed
that the fibers were attached and had melted into each
other. Variation in fibers for each sample also changed in
cases of PLGA/PCL (20/80), the diameter for over 60% of
the fibers was around 500 ± 100 nm. For the PLGA/PCL
(10/90) electro-spun fibers, the diameter for over 60% of
the fibers was around 1,000 ± 200 nm (Fig. 1a). Finally,
J Mater Sci: Mater Med
the PLGA/PCL (30/70) electro-spun fibers were attached to
each other and the diameter for over 50% of the fibers were
around 2,000 ± 200 nm and the remaining fibers were
very large.
3.2 FT-IR
To determine if the blended PLGA/PCL electro-spun fibers
were successfully fabricated, we analyzed the FT-IR
spectrum of PCL, PLGA, and PLGA/PCL at different
PLGA/PCL ratios (Fig. 2). The FT-IR spectrum of the
blend PLGA/PCL electro-spun fibrous mats showed that
the structure of blend electro-spun mats contained all the
peaks corresponding to PCL and PLGA. More specifically,
all of the PCL peaks in the PLGA/PCL composite over-
lapped with all of the PLGA peaks without the peak at
865 cm-1, which appeared separate from the PCL peaks.
In this case, the PLGA peaks had large widths but not
depths while the PCL peaks had large depths but not
widths. In addition, the amount of PLGA was smaller than
the amount of PCL. Therefore, a larger PCL width in the
blend electro-spun PLGA/PCL fibers was expected. Fig-
ure 2 also demonstrates that no reaction between PCL and
PLGA occurred; these fibers were just blended composites.
Almost peaks of the blend PLGA/PCL electro-spun fibrous
mats overlapped PCL peaks, it is easy to confuse but the
peaks at 865 cm-1 which asserted PLGA present. How-
ever, the intensity of this peak increased with increasing
amounts of PLGA. The combined results demonstrate that
the blend PLGA/PCL electros-pun fibrous mats were suc-
cessfully fabricated.
3.3 Mechanical property
Since a single polymer nano-fiber is very weak, the
resulting non-woven mat was so delicate that any direct
Fig. 2 FT-IR curves of electrospun PLGA (a), PCL (b), PLGA/PCL
(10/90) (c), (20/80) (d), (30/70) (e)
contact with the mat surface during manipulation could
damage the fibers. Therefore, the samples were collected
using alumina foil at a similar polymer concentration, and
then the thickness of each sample was measured by SEM.
These samples were measured, cut carefully and glued on
the frame (Fig. 3), and then the aluminum foil was
removed. The paper frame was cut before initiating the
tensile strength measurements. The tensile strengths of the
electro-spun PLGA/PCL fiber mat at different PLGA
concentrations were measured using a tensile tester (Uni-
versal Testing Machine, R&D Co., Ltd., Korea). Figure 4A
shows the curves of PCL and the PLGA/PCL composites at
different PLGA concentrations. In these experiments, we
found that the stress in the electro-spun PLGA/PCL elec-
tro-spun mats increased as the amount of PLGA increased.
However, the strain did not always increase following an
increase in the amount of PLGA. In the case of PCL
(Fig. 4Aa) and PLGA/PCL (10/90) (Fig. 4Ab), the stress
increased from 1.8 ± 0.05 to 2.5 ± 0.05 MPa and the
strain increased from 130 ± 5 to 134 ± 5 (%). However,
this same trend was not observed when the PLGA con-
centration was increased from 10% [PLGA/PCL (10/90)]
(Fig. 4Ab) to 20% [PLGA/PCL (20/80)] (Fig. 4Ac). In this
scenario, the stress increased from 2.5 ± 0.5 MPa to
6.1 ± 0.5 MPa but the strain decreased from 134 ± 5 to
88 ± 5 (%). When the PLGA concentration was increased
Fig. 3 A schematic of the paper frame used to prepare the tensile
specimens from the electro-spun non-women mats
Fig. 4 A Stress–strain curves of electrospun PCL (a), PLGA/PCL
(10/90) (b), PLGA/PCL (20/80) (c), PLGA/PCL (30/70) (d). B
Variation of tensile strength of PCL and PLGA/PCL blend
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 J Mater Sci: Mater Med

from 20% [PLGA/PCL (20/80)] (Fig. 4Ac) to 30% [PLGA/

PCL (30/70)] (Fig. 4Ad) the strain and stress both suddenly
decreased from 6.1 ± 0.5 to 3.8 ± 0.5 MPa and 88 ± 5
(%) (strain) to 59 ± 5 (%), respectively. The variations in
tensile strength, which contains more detail on the rela-
tionship between the stress and strain of each mat, is shown
in Fig. 4B. As shown in Fig. 4Ad, the stress and strain
were not high even though the amount of PLGA was high.
These results most likely occurred because the electro-spun
fibers containing 30% PLGA were not fine as the PLGA/
PCL (20/80) and PLGA/PCL (10/90) fibers. In addition,
PLGA is a very brittle material [15], which can easily
break if the stress increases. Duan et al. [8]. reported a Fig. 5 MTT cytotoxicity of the blend electro-spun PLGA/PCL mats
stress of 9 MPa and strain of 30% for only PLGA elec- at different PLGA/PCL ratio
trospun mats. For this reason, the mechanical properties of
the electrospun PLGA/PCL mats increased at higher
amounts of PLGA. Based on the combined results pre- 3.5 Cellular proliferation
sented above, we concluded that the stress of the PLGA/
PCL fibers was dependent on both the amount of PLGA The proliferation of fibroblast cells on the blend electro-
and the uniformity of the electro-spun fibers on the mats. spun PLGA/PCL fibrous mats was evaluated by the
Because the fibers of the electro-spun PLGA/PCL (30/90) reduction of MTT to formazan by mitochondrial succinate
aggregated with each other and the composite was not dehydrogenase in complex II (succinate–ubiquinone oxi-
uniform, the stress was lower than the PLGA/PCL (20/80) doreductase complex) [23]. The level of reduction of
mats. The co-polymer containing 10 wt% PLGA had a MTT into formazan reflects the level of cell metabolism.
strain value that was higher than the composites not con- Figure 6 compares the time course of formazan absor-
taining PLGA. The stress–strain data showed that PCL was bance of blend PLGA/PCL nanofiber mats fabricated with
a flexible polymer and if it was added to brittle PLGA different concentrations of PLGA. Biomimetic tissue
materials (non-flexible polymer) in small amounts, the formed on the blend PLGA/PCL nanofibrous mats, which
mechanical strength under fine fibrous mats conditions was affected the viability and proliferation of cells. Cell
increased. attachment and proliferation on the electro-spun PLGA/
PCL (10/90) co-polymer doubled between day 1 and day
3.4 Cytotoxicity results 3. Moreover, the absorbance values showed that cell
attachment and proliferation increased with an increase
The cytotoxicity of PLGA/PCL nanofiber mats was tested of PLGA concentration in the PLGA/PCL composite;
by quantitative analysis using the MTT test. After allowing however, between PLGA/PCL (20/80) and PLGA/PCL
the cells to spread for 3 days at the various extract dilutions (30/70), a slight increase was observed. According to
(0, 25, 50, 75 and 100%), the proliferation of L-929 cells the results, we concluded that electro-spun PLGA/PCL
was measured by the MTT assay. In the case of the control, (20/80) had the greatest effect on ECM production when
the extract solution containing cultured cells was not compared to PLGA/PCL (30/70) and PLGA/PCL (10/90)
added. Absorbance values were used to measure cellular (Fig. 6).
proliferation. The extracts containing the blend electrospun
PLGA/PCL nanofiber mats did not inhibit cell metabolism
relative to the control group. We found that cell metabo-
lism of the blend electro-spun PLGA/PCL nano-fibrous
increased with an increase in the PLGA concentration.
Based on the quantitative scores, it was concluded that the
extracts of the nanofibers mats displayed no cytotoxic
reactivity in this test, as shown in Fig. 5. In addition, nei-
ther cell death nor morphological disorder was observed
throughout the incubation period. Thus, it was concluded
that PLGA/PCL nano-fibrous mats produced from this
process displayed no cytotoxic effect due to the presence of Fig. 6 Cellular proliferation of the blend electro-spun PLGA/PCL of
remnant solvents. fibroblast cells L-929 after seeding the cells for 1 day and 3 days

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J Mater Sci: Mater Med
3.6 Cell attachment and cell proliferation
3.6.1 One cell morphology-cell attachment
The cell morphology was examined to continually check
the biocompatibility of all PLGA/PCL blend membranes.
Figure 7 shows the cell morphology of one fibroblast
L-929 after seeding the cell for 1 h on a tissue culture plate
(TCPs) (Fig. 7a1–a3). Figure 7a2, a3 shows an image of
cell attachment on the PLGA/PCL (10/90) electro-spun
mat, while Fig. 7b2, 3 shows an image of cell attachment
on the PLGA/PCL (20/80) electro-spun mat. Finally,
Fig. 7c2, c3 shows an image of cell attachment on the
PLGA/PCL (30/70) electro-spun mat. By comparing the
filopodium and lamellipodium in the control sample with
each condition, we found that filopodium and lamellipo-
dium growth was as good as the control. However, by
comparing (Fig. 7a2, b2, c2), we found that filopodium and
lamellipodium growth increased as the content of PLGA
increased. On the other hand, in the enlarged image
Fig. 7 Cell morphology of fibroblast cell growth on control (a1, b1, c1), blend PLGA/PCL (10/90) (a2, a3), PLGA/PCL (20/80) (b2, b3) and
PLGA/PCL (30/70) (c2, c3) electro-spun after seeding cell 1 h
(Fig. 7a3, b3, c3), which shows more detail, the L-929
cells were shown to be well attached to the PLGA/PCL
fiber. Therefore, the PLGA/PCL electro-spun fibrous
has high biocompatibility that is suitable for biomaterial
applications.
3.6.2 Cell proliferation
To test cell proliferation on the PLGA/PCL electro-spun
fibrous mats, we seeded the cells at the same density and
incubated the samples for different time periods (short-
time). Figure 8 shows the cell proliferation after seeding
the cells for 1 day, while Fig. 9 shows the cell proliferation
after seeding the cells for 3 days. After 1 and 3 days of cell
seeding the cells were spread on all scaffolds. In Fig. 8, the
images showed that the fibroblast cells were spread on the
TCP and PLGA/PCL fibrous mats after seeding the cells
for 1 day. These results demonstrated cellular attachment
and proliferation of mouse fibroblast L-929 cells under
static seeding on all PLGA/PCL electrospun mats.
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Fig. 8 SEM morphology of fibroblast cells grown on the control (a), blend PLGA/PCL (10/90) (b), PLGA/PCL (20/80) (c) and PLGA/PCL
(30/70) (d) electro-spun after seeding cell 1 day
However, cell proliferation on the samples [Fig. 8b–d] was
clearly better than the TCP surface [Fig. 8a] as evidenced
by the development of lamellopodia, which is an indicator
of cell attachment to the surface. In addition, after 1 day of
seeding, cells were well grown and had spread uniformly
and extensively, covering the surfaces of the biomaterials,
which was similar to the control sample. The presence of
lamellapodia and an interlaced fibrous network demon-
strated that cell adhesion was very good. Cell spread-
ing increased on the PLGA/PCL electrospun mats when
the PLGA concentration was increased (Figs. 8b and
8 [c, d]).
After 3 days (Fig. 9), cells completely covered the
electro-spun mats. To assess cell proliferation and cells
attachment on the surface, we took images of each sample at
different magnifications. At the highest magnification
(Fig. 9a3, b3, c3), we found that cell proliferation increased
at higher PLGA concentrations. In addition, cell attachment
was more clearly observed on the PLGA/PCL (30/70)
electro-spun fibrous mat. Even though the incubation time
was just 3 days, the shape of the fibroblast cells changed
from round to elongated (Fig. 9a3, b3, and c3). Based on
these results, we can conclude that the electro-spun PLGA/
123
J Mater Sci: Mater Med
PCL co-polymer is a good composite for tissue engineering
applications.
4 Discussion
In this study, we investigated a new component for
biomaterials and tissue engineering applications; the elec-
tro-spun PLGA/PCL blend. We fabricated PLGA/PCL
electro-spun mats by combining different PLGA and PCL
ratios into a single PLGA/PCL component. Calvert et al.
[24] and Lee et al. [25] reported that PCL should not be used
at high percentages in PLGA/PCL composites due to its
high cost and stability during handling and storage [14].
Therefore, we combined PCL with different ratios of PLGA
to fabricate PLGA/PCL electro-spun fibrous mats. On the
other hand, Calvert et al. [24] also reported that a high
percentage of PLGA was not always as good as a low
percentage; therefore, we only used a maximum PLGA
concentration of 30% in the PLGA/PCL blend. The SEM
morphology shown in Fig. 1 clearly demonstrates that
PLGA concentration was very important in obtaining fine
PLGA/PCL fibers mats. The PLGA/PCL electro-spun fiber
J Mater Sci: Mater Med
Fig. 9 SEM morphology of fibroblast cells grown on blend PLGA/PCL (10/90) (a1, a2, a3), PLGA/PCL (20/80) (b1, b2, b3) and PLGA/PCL
(30/70) (c1, c2, c3) electro-spun after seeding the cells for 3 days
for PLGA/PCL (10:90) was very large and its mechanical
strength was much higher than the composite not containing
PLGA. However, at 20% PLGA, the PLGA/PCL electro-
spun blend contained very fine fibers with small, smooth,
cylindrical, and humongous fibers, at this composition the
stress of the electro-spun mat was very high but the strain
was lower relative to the PLGA/PCL (10/90) electro-spun
mat. As for the PLGA/PCL (30/70) composite, the high
PLGA concentration changed the heat of fusion, which
affected the blend solution, resulting in a morphology that
showed a blend of PLGA/PCL (30/70) electro-spun fibers
were larger. The morphology of PLGA/PCL (30/70) elec-
tro-spun mat is a key to make the mechanical strength the
PLGA/PCL (30/70) electro-spun mats were lower than the
PLGA/PCL (20/80) electro-spun mat. Lee et al. [25] fab-
ricated PCL electro-spun nonwoven mats from PCL in a
DMF: MC (25/75) solution, which displayed a strain of over
200% and stress of approximately 1.7 MPa. In this study,
the PCL electro-spun mat was fabricated from PCL in a
DMF: MC (80/20) solution and had a similar stress but
lower strain (130%). The PLGA/PCL (20/80) electro-spun
mat showed the best mechanical strength and modulus
strength because this fibrous mat was uniform and small in
diameter, both of which are important factors dictating the
mechanical properties of electrospinning [7, 26]. The FT-IR
curves showed that PLGA/PCL electro-spun mats were
successfully fabricated using the PLGA/PCL blend due to
the presence of the 865 cm-1 peak. The PLGA/PCL elec-
tro-spun mat was shown to not be toxic at all compositions
(Fig. 5). In addition, cell proliferation of fibroblast doubled
between day 1 and day 3 day (Fig. 6) through MTT results.
On the other hand, the cells became attached to the PLGA/
PCL electro-spun mats within 60 min (Fig. 7). The SEM
images showed that filopodium and lamellipodium growth
on the electro-spun mats was as good as the control. But, the
cell proliferation and attachment was better as the PLGA
concentration in the PLGA/PCL electro-spun mats was
increased after seeding the cells for 1 day (Fig. 8) and
3 days (Fig. 9). However, in the case of PLGA/PCL (30/
70), the cells did plate on the electro-spun mat but the cells
shrunk. These combined results clearly indicate that the
presence of the PLGA support in the PLGA/PCL electro-
spun mats improved biocompatibility, cell attachment and
cell proliferation.
123

5 Conclusion
In this study, we successfully fabricated electro-spun
PLGA/PCL mats containing different concentrations of
PLGA. PCL was used as the major material because it is
inexpensive and stable during handling and storage. PCL is
a flexible material and PLGA is a brittle material; thus, the
PLGA/PCL electro-spun mats had a stronger mechanical
strength than PCL. The addition of PLGA as a doping
agent in the PLGA/PCL electro-spun mats increased the
value of this composite for biomaterial and tissue engi-
neering applications because it increased biocompatibility,
cell attachment and cell proliferation. We investigated a
new electro-spun component and demonstrated that it holds
promise for use as a biomaterial for tissue engineering and
biomedical applications.
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